The Escherichia coli uvr ABC system catalyzes the incision of damaged DNA. Having available reagent quantities of homogeneous UurB and UvrC proteins permits a detailed examination of the individual partial reactions leading to the dual incision stage of nucleotide excision repair. The pre-incision steps can be subdivided into a number of partial reactions which include: (a) UvrA dimerization stimulated by ATP binding, (b) UvrA-nucleoprotein formation at both damaged and undamaged sites, (c) the accompanying topological unwinding stimulated by ATP binding, (d) the participation of the UvrB cryptic ATPase in the UvrAB catalyzed strand displacement reaction and finally (e) dual incision catalyzed by the presence of UvrC. The incision mechanisms precede the multi-nucleoprotein complex requiring coordinated excision reactions catalyzed by UvrD, DNA polymerase I and polynucleotide ligase. In the principal direction for the proposed studies we will attempt to associate the anatomy, or structure of the respective uvr A and B genes to the catalytic and protein properties of the related gene product proteins. The focal points and role of ATP in the individual processes will also be addressed. The protein sequences, or domains, of interest include putative ATP binding regions, sites sensitive to a protease specific for the Ada protein of E. coli potential DNA binding sites and to a limited extent the "zinc finger"-like sites. These sites will be engineered by oligonucleotide-directed and deletion mutants, the individual clones sequenced and over-expressed in suitable expression vectors and the catalytic and protein properties of the mutant and "wild type" proteins examined for their enzymatic phenotypes in the respective pre-, post- and incision steps. The biological role and biochemical nature of UvrB proteolysis in regulation of repair will be further investigated by genetic, structural and catalytic methods.